Experimental Diab. Res., 4:73–81, 2003Copyright c© Taylor and Francis Inc.ISSN: 1543-8600 print / 1543-8619 onlineDOI: 10.1080/15438600390220016

Growth Hormone (GH) Hypersecretion and GH ReceptorResistance in Streptozotocin Diabetic Mice in Responseto a GH Secretagogue

Peter B. Johansen,1 Yael Segev,2 Daniel Landau,2 Moshe Phillip,2and Allan Flyvbjerg3

1Pharmacological Research 3, Novo Nordisk A/S, Måløv, Denmark2Molecular Endocrinology Laboratory, Department of Pediatrics, Soroka University Medical Center,Beer-Sheva, Israel3Medical Department M/Medical Research Laboratory, Institute of Experimental Clinical Research,Aarhus Kommunehospital, Aarhus, Denmark

The growth hormone (GH) and insulin-like growth fac-tor I (IGF-I) axis were studied in streptozotocin (STZ) di-abetic and nondiabetic female mice following intravenous(IV) injection of the GH secretagogue (GHS) ipamorelin orsaline. On day 14, blood samples were obtained before and10 minutes after the injection. Livers were removed andfrozen for determination of the mRNA expressions of theGH receptor, GH-binding protein, and IGF-I, and hepaticIGF-I peptide. Serum samples were analyzed for GH andIGF-I. Following ipamorelin injection, the GH levels werefound to be 150 ± 35 µg/L and 62 ± 11 µg/L in the diabeticcompared to the nondiabetic mice (P < .05). Serum IGF-Ilevels were lower in diabetic than in nondiabetic animals,and rose after stimulation only in the nondiabetic animals.Furthermore, hepatic GH resistance and IGF-I mRNA lev-els and IGF-I peptide were increased in nondiabetic animalsin response to GH stimulation, whereas the low levels per seof all these parameters in diabetic mice were unaffected. The

Received 31 July 2002; accepted 24 February 2003.This study was supported by grants from the Danish Medical Re-

search Council (no. 9700592), the Novo Foundation, the Aage Louis-Hansen Memorial Foundation, the Nordic Insulin Foundation, the Evaand Henry Frænkels Memorial Foundation, and the Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration (no.9600822). The authors are grateful to Mrs. Karen Mathiassen, KirstenNyborg, and Ninna Rosenqvist for excellent technical assistance.

Address correspondence to Peter B. Johansen, PhD, Pharmacolog-ical Research 3, Novo Nordisk A/S, Novo Nordisk Park, DK-2760Måløv, Denmark. E-mail: pjoh@novonordisk.com

study shows that STZ diabetic mice demonstrate a substan-tial part of the clinical features of type 1 diabetes in humans,including GH hypersecretion and GH resistance. Accord-ingly, it is proposed that STZ diabetic mice may be a bettermodel of the perturbations of the GH/IGF-I axis in diabetesthan STZ diabetic rats.

Keywords Diabetes; Growth Hormone Resistance; GrowthHormone Secretion; Insulin-Like Growth Factors;Ipamorelin; Mice; Streptozotocin

Growth hormone (GH) and insulin-like growth factors(IGFs) have a long history in diabetes mellitus, with conceivableeffects on the development of diabetic renal complications; forreview, see [1–4]. Poorly controlled type 1 diabetes in man ischaracterized by GH hypersecretion and low circulating IGF-Ilevels [5–8]. The streptozotocin (STZ) diabetic rat, however,is characterized by suppressed serum GH levels and loss ofthe characteristic pulsatility [9–11]. It has been reported thatpituitary GH concentration is significantly reduced in STZ dia-betic rats as compared to that observed in control animals. Thereduced GH levels are probably due to a deficiency in GH stor-age and/or synthesis [12]. Both increased [13] and decreased[14] GH levels have been found in the Biobred (BB) rat, whichis considered to be the classical model of early (autoimmune)onset type 1 diabetes.

The highly conspicuous species difference in neuroregula-tion of the GH axis in diabetes is also present during other

73

74 P. B. JOHANSEN ET AL.

exogenous stimuli, such as exercise, starvation, and stress; forreview, see [15]. To be able to extent results obtained in ani-mal studies into humans, it is imperative that the animal modelclosely reflects the clinical condition. In the present paper, weshow enhancement of GH secretion also in STZ diabetic micefollowing intravenous (IV) injection of the GH secretagogue(GHS) ipamorelin.

MATERIALS AND METHODS

Animals and ProtocolFour groups of adult female Balb/C(a) mice (Bomholtgaard,

Ry, Denmark), with initial body weights of 15 to 19 g, werestudied. The mice were housed 6 to 8 per cage in a room with12:12-hour light cycle, temperature 21◦C ± 2◦C and humid-ity 55% ± 2%. The animals had free access to standard chow(Altromin no. 1324, Lage, Germany) and tap water through-out the experiment. The animals were randomly allocated to4 groups, and 2 of these groups were made diabetic by a sin-gle IV injection of STZ (Upjohn Company, Kalamazoo, MI,USA), in a dose of 300 mg/kg body weight. The developmentof diabetes was followed over the next 14 days by measur-ing blood glucose by Haemoglucotest 1-44 and Reflolux II re-flectance meter (Boehringer-Mannheim, Mannheim, Germany)and urine for glucose and ketone bodies by Neostix-4 (Ames,Stoke Poges, Slough, UK). Only animals with blood glucoseabove 16 mmol/L, glucosuria above 111 mmol/L, and withoutketonuria were included in the study. Body weight and foodconsumption were recorded.

At day 14, ipamorelin, 200 µg/kg, or saline, 5 mL/kg, wereinjected intravenously. Blood was drawn before and exactly 10minutes after the injection from the retro-orbital venous plexusfor determination of serum GH, IGF-I, and IGF-binding protein3 (IGFBP-3). Serum was stored at −80◦C until measurementswere performed. Furthermore, the kidneys and livers wererapidly removed and carefully cleaned and weighed. The liverswere frozen in liquid nitrogen and stored at −80◦C for laterdetermination of the mRNA of the GH receptor (GHR), GH-binding protein (GHBP), and IGF-I, and hepatic IGF-I peptide.

The study was performed under an approved animal per-mit complying with national regulations for use of animals inresearch (the Danish Animal Inspectorate).

ImmunoassaysSerum GH was measured by radioimmunoassay (RIA) us-

ing a specific polyclonal rabbit rat(r) GH antibody and rGHas standard. Semilog linearity of mouse serum and rGH (inthe standard) was found at multiple dilutions, indicating anti-gen similarity between mouse GH and rGH. The ingredients,including [125I]-rGH, were obtained from Amersham Interna-

tional, Bucks, UK. Serum IGF-I was measured after extractionwith acid-ethanol. The mixture was incubated for 2 hours atroom temperature, centrifuged, and 25 µL of the supernatantwas diluted 1:200 before analysis. Liver extraction was per-formed as previously described [16]. Briefly, 80 to 100 mgof liver tissue was homogenized on ice in 1 M acetic acid(5 mL/g tissue) with an Ultra Turrax TD 25 and further dis-rupted using a Potter Elvehjelm homogenizer. An additionalextraction procedure (with ethanol/HCl) was performed in liverhomogenates to fully remove all IGFBPs. After lyophilization,the samples were redissolved in phosphate buffer (pH 8.0) andkept at −80◦C until the IGF-I assay was performed in dilutedextracts. Serum and liver IGF-I levels were measured by RIAusing a polyclonal rabbit antibody (Nichols Institute Diagnos-tics, San Capistrano, CA, USA) and recombinant human IGF-Ias standard (Amersham International). The tissue IGF-I concen-trations were corrected for the contribution of entrapped serumIGF-I [16]. Monoiodinated IGF-I ([125I-Tyr31]-IGF-I) was ob-tained from Novo Nordisk A/S, Bagsværd, Denmark. Intra- andinterassay coefficients of variations for both assays were

GH HYPERSECRETION IN STZ DIABETIC MICE 75

TABLE 1Body weight, blood glucose, food consumption, kidney weight (both kidneys), and liver weight in nondiabetic mice injected

with saline (C/SAL) or ipamorelin (C/IPA) and diabetic animals injected with saline (D/SAL) or ipamorelin (D/IPA) at day 14

Body weight Blood glucose Food consumption Kidney weight Liver weight(g) (mM) (g/day) (mg) (mg)

C/SAL 20.2 ± 0.4 6.1 ± 0.4 5.4 ± 0.9 245 ± 3 1178 ± 46C/IPA 19.9 ± 0.3 5.9 ± 0.5 5.9 ± 0.5 244 ± 6 1074 ± 51D/SAL 15.2 ± 0.3∗ 22.8 ± 0.7∗ 9.2 ± 0.8∗ 269 ± 6∗ 898 ± 38∗D/IPA 15.5 ± 0.4∗ 23.8 ± 1.0∗ 9.6 ± 0.9∗ 264 ± 6∗ 902 ± 76∗

Note. Values are means ± SEM, n = 7–10 per group.∗P < .01 versus respective control groups (Student’s t test for unpaired comparisons).

quantified by absorbency at 260 nm. The integrity of the RNAwas assessed by visual inspection of the ethidium bromide–stained 28S and 18S RNA bands after electrophoresis through1.25%/2.2 M formaldehyde gels. For Northern blot analysis,20 µg of total RNA were electrophoresed on 1.3% agarose/2.2 M formaldehyde gels in 3-morpholinopropanesulphonicacid buffer. The RNA was then transferred onto Magna-Graph (MSI, Westboro, MA, USA) nylon membranes andwas cross-linked to the membrane with an ultraviolet (UV)cross-linker (Hoefer Scientific Instruments, San Francisco, CA,USA). A rat GHR/GHBP probe (a generous gift from L.Mathews) was radiolabeled with [32P]-dCTP (Amersham Inter-national) by a random-primed DNA labeling kit (Boehringer-Mannheim). RNA hybridization was performed in a hybridiza-tion oven (Micro-4; Hybaid Ltd, UK) at 65◦C for 20 hoursin a hybridization solution (0.2 mM Na2HPO4, pH 7.2, 7%[vol/vol] SDS, 1% [wt/vol] BSA, and 1 mM EDTA). Gelswere exposed to Kodak X-Omat AR film at −70◦C withtwo intensifying screens. The autoradiograms were quan-tified using a phosphoimager (Imagequant; Molecular Dy-namics, Sunnyvale, CA, USA). IGF-I mRNA levels weredetermined using a solution hybridization/RNase protectionassay. In brief, 25 µg of total RNA was hybridized with400,000 cpm of 32P-labeled riboprobe for 16 hours at 45◦Cin 75% formamine–0.4 M NaCl, followed by digestion with40 g/L RNase T1. Protected hybrids were precipitated, dena-tured, and electrophoresed through 8% urea–polyacrylamidegels. Gels were exposed to Kodak X-Omat AR film as de-scribed above, and the protected band (231 bp) on the autora-diography that corresponds to IGF-I was analyzed. The autora-diograms were quantified using a phosphoimager as describedabove.

StatisticsStudent’s t test for unpaired comparisons was used. A P

value of less than 5% was regarded as significant. Means ±SEM are given.

RESULTS

Body Weight, Metabolic Parameters, and FoodConsumption

At day 14, the STZ-treated mice were markedly diabetic,with blood glucose levels 4-fold higher than those of the con-trol mice. Also, food consumption and kidney weight were in-creased, and liver weight was decreased in the STZ diabeticmice (Table 1).

Serum GH, IGF-I, and IGFBP-3No differences were observed in the basal GH levels. Follow-

ing GHS stimulation, however, the serum GH levels were foundto be 150 ± 35 and 62 ± 11 µg/L, respectively, in the diabeticcompared with the nondiabetic animals (P < .05) (Figure 1).Differences in the basal serum IGF-I levels were observed be-tween the diabetic and nondiabetic animals, being 208 ± 23 and418 ± 22 µg/L, respectively (P < .0001). After stimulation, theserum IGF-I level did not change in the diabetic treated ani-mals, but increased to 522 ± 30 µg/L in the nondiabetic animals(P < .01).

A representative autoradiogram of the IGFBP-3 WLB isshown in Figure 2A. The IGFBP-3 levels were lower in dia-betic as compared to nondiabetic animals, but ipamorelin stim-ulation did not induce any changes neither in diabetic nor innondiabetic animals (Figure 2B).

Hepatic GHR/GHBP mRNA, IGF-I mRNA,and IGF-I Peptide

Autoradiograms of the ethidium bromide staining of theRNA samples before their transfer to the membrane and theNorthern blot analysis for the GHR and GHBP mRNAs areshown in Figures 3A and 3B, respectively. Despite the fact thatthe basal GH levels were comparable in diabetic and nondia-betic animals, a significant lower GHR mRNA expression wasfound in the diabetic animals. After ipamorelin injection, theGHR mRNA level rose significantly, but only in the nondiabetic

76 P. B. JOHANSEN ET AL.

FIGURE 1Serum growth hormone (GH) concentrations (µg/L) in nondiabetic mice injected with saline (C/SAL) or ipamorelin (C/IPA)

and diabetic animals injected with saline (D/SAL) or ipamorelin (D/IPA) 10 minutes after injection at day 14. Values aremeans ± SEM, n = 7–8. #P < .05 compared with C/IPA; ++P < .01 compared with C/SAL. Student’s t tests for unpaired

comparisons were used.

animals. Also, GHBP mRNA levels were lower in diabeticanimals, but the expression was unchanged by ipamorelin stim-ulation. IGF-I mRNA expression and hepatic IGF-I peptideconcentrations were increased by ipamorelin stimulation inthe nondiabetic animals, whereas no changes were induced inthe diabetic animals. The quantitative Northern blot analysis isshown in Figure 4.

DISCUSSIONPoorly controlled type 1 diabetes in humans is characterized

by GH hypersecretion [6, 8], whereas GH secretion in diabeticrats is decreased [10, 11]. There is, on the other hand, supportfor the contention that the difference between experimental di-abetes in rats and human diabetes, with respect to the GH/IGFaxis, is restricted to GH. Accordingly, identical changes havebeen reported for the other elements in the axis in diabetic ratsand man, including changes in circulating levels of GHBP, IGF-I, and IGFBPs; for review, see [19]. Also, disturbed hepatic and

renal transcriptional changes and decreased hepatic GHR num-ber are well-described features in experimental diabetes in rats[20–23].

The major new finding of the present study is the demon-stration of GH hypersecretion in STZ diabetic mice after a GHstimulation test using the GHS ipamorelin. After GHS stimula-tion, a 2- to 3-fold increase in serum GH levels was evident in thediabetic mice when compared to the nondiabetic control mice.The sampling time was chosen from studies in rats and micewhere the systemic GH levels were shown to peak 10 minutesafter IV injection of ipamorelin [24, 25]. Ipamorelin belongs toa class of peptide compounds that have been demonstrated topossess a dual action at the pituitary as well as the hypothalamiclevels. Thus, GHS provokes the release of GHRH into hypophy-seal portal blood [26], and it was recently reported that passiveimmunization with anti-GHRH serum almost completely oblit-erated the GH response to GHRP-6 [27]. These data clearlydemonstrate that the vast majority of the GH secretion afterGHS stimulation in vivo is caused by the release of GHRH.

GH HYPERSECRETION IN STZ DIABETIC MICE 77

(A)

(B)

FIGURE 2(A) Representative Western ligand blot (WLB) autoradiogram of serum samples from nondiabetic mice injected with saline

(C/SAL) (lanes 1 and 2) or ipamorelin (C/IPA) (lanes 5 and 6) and diabetic mice injected with saline (D/SAL) (lanes 3 and 4) oripamorelin (D/IPA) (lanes 7 and 8). Serum IGFBP-3 appears as a 38- to 42-kDa doublet band corresponding to the intact

acid-stable IGF-binding subunit of IGFBP-3. Mr, molecular weight. (B) Quantitative analysis of serum insulin-like growthfactor I (IGF-I) concentrations (µg/L) and IGF-binding protein 3 (IGFBP-3) levels (pixel) in nondiabetic mice injected with

saline (C/SAL) or ipamorelin (C/IPA) and diabetic animals injected with saline (D/SAL) or ipamorelin (D/IPA) 10 minutes afterinjection at day 14. Values are means ± SEM, n = 7–8. ∗P < .02 compared with C/SAL; +P < .0001 compared with C/SAL;

++P < .0001 compared with C/IPA. Student’s t tests for unpaired comparisons were used.

78 P. B. JOHANSEN ET AL.

(A)

(B)

FIGURE 3(A) Ethidium staining of the RNA samples before their transfer to the membrane. The locations of the 28S and 18S ribosomal

RNA bands are depicted. (B) Northern blot analysis of liver total RNA (20 µg) from nondiabetic mice injected with saline(C/SAL) (lanes 1 to 3) or ipamorelin (C/IPA) (lanes 4 to 7) and diabetic mice injected with saline (D/SAL) (lanes 8 to 10) or

ipamorelin (D/IPA) (lanes 11 to 14). The 4.4-kb and 1.2-kb bands corresponding to the GHR and GHBP are shown on the left.

The elevated stimulated serum GH in diabetic mice is con-trary to previous reports on the usual model of animal type 1diabetes, the STZ diabetic rat. In this animal, decreased GH lev-els have been described, probably due to the inhibition of GHrelease from the pituitary [11]. It should be noted, however,that the diabetic state in the present study and in all studies in-vestigating GH secretion in rats have used a single high-doseSTZ injection. Induction of type 1 diabetes in rats or mice canalso be carried out by repeated daily low-dose STZ injections,which are less likely to give kidney toxicity and ketosis-prone

diabetes. In the present study, however, the mice had a sta-ble B-glucose without signs of ketosis. It has previously beendemonstrated that a single high-dose STZ injection does notinflict kidney toxicity per se, except for the well-known kidneychanges caused by the diabetic state [16]. Whether a differencein GH affection exists between the 2 STZ regimens remains tobe shown.

The stimulated GH levels of the nondiabetic mice wereaccompanied by increases in circulating and hepatic IGF-Ipeptide and mRNA levels. These changes were absent in the

GH HYPERSECRETION IN STZ DIABETIC MICE 79

FIGURE 4Quantitative analysis of hepatic GHR, GHBP, IGF-I mRNA (pixel), and hepatic IGF-I peptide (ng/g) levels in nondiabetic miceinjected with saline (C/SAL) or ipamorelin (C/IPA) and diabetic animals injected with saline (D/SAL) or ipamorelin (D/IPA)

10 minutes after injection at day 14. Values are means ± SEM, n = 3–4. ∗P < .002 compared with C/SAL; +P < .0002compared with C/SAL; ++P < .0001 compared with C/IPA; #P < .05 compared with C/SAL; §P < .0005 compared with C/IPA.

Student’s t tests for unpaired comparisons were used.

diabetic animals. Traditionally, the majority of circulating IGF-Iis synthesized in the liver, mainly under the control of circu-lating GH, and thus in hyperglycemic diabetic subjects, serumIGF-I is reduced in spite of elevated GH. In order to explainthis phenomenon, it has been hypothesized that the diabeticmetabolic deterioration first decreases hepatic IGF-I formationand serum IGF-I levels, which then secondarily induces GH hy-persecretion [1, 3, 5–8]. This hypothesis is in accordance withthe findings of the present study, and is in agreement with re-sults obtained in recent studies in diabetic mice [3, 16, 28]. GHreceptor resistance has been suggested by previous clinical andexperimental studies in type 1 diabetes. The previous findingsinclude (a) decreased liver GHBP in diabetic states; (b) reducedplasma concentrations of GHBP in untreated spontaneously di-abetic BB rats with full normalization during insulin adminis-

tration [23]; (c) decreased serum GHBP in human patients withinsulin-dependent diabetes mellitus (IDDM) [29]; and (d) re-duced IGF-I secretion associated with low hepatic GHR levels,which also normalize during insulin treatment [30].

To our knowledge, only one study has addressed the GHprofile in conscious STZ diabetic mice fitted with indwellingcatheters [31]. In this study, suppression of episodic GHsecretion was reported. The pituitary GH responses in vitro todifferent doses of GHRH, however, were found to be remark-ably increased, suggesting an up-regulation of the pituitaryGHRH receptor caused by a decrease in hypothalamic GHRHsecretion. Also, the pituitary GH content was reduced. If thereduced GH levels in vivo were caused by decreased GHRHsynthesis/release, the enhanced GH secretion following GHSstimulation in the present study is difficult to explain, because

80 P. B. JOHANSEN ET AL.

the GH release following GHS stimulation is predominantlyconveyed by GHRH. One conspicuous difference between the2 studies was found in the blood glucose levels (32 versus24 mmol/L), suggesting that the diabetic aberration had beenmore severe in the study by Murao and colleagues [31]. Itcan be speculated that the differences in GH levels are due todifferences in diabetic aberration. Of note, no differences inthe basal GH levels were observed in the present study. Thisis contrary to the findings in nonobese diabetic (NOD) mice[28].

Recent studies have described renoprotective effects of GHantagonists in long-term diabetic transgenic mice that expressthe GH antagonists bGH-G119R and hGH-G120R [16, 32, 33].Also, normalization of diabetic renal and glomerular hypertro-phy, along with a reduction in the diabetes-associated increasein urine albumin excretion, have been reported in diabetic micetreated with the GH antagonist G120K-PEG [16, 34]. Theseobservations are in agreement with the present finding of GHhypersecretion in STZ diabetic mice and that GH (and IGFs)is involved in the pathogenesis of diabetic kidney disease inexperimental diabetes.

In conclusion, the present paper reports enhanced GH secre-tion in STZ diabetic mice when compared to nondiabetic micefollowing stimulation with a GH secretagogue. Consequently,the STZ diabetic mouse seems to reflect better the perturbationsof the circulating GH/IGF levels as seen in human type 1 dia-betes, and may be a more suitable model than the STZ diabeticrat. As only few studies on GH secretion in STZ diabetic micehave been published, more extended studies on this topic arehighly warranted.

REFERENCES[1] Flyvbjerg, A. (1990) Growth factors and diabetic complications.

Diabetic Med., 7, 387–399.[2] Flyvbjerg, A., Froesch, ER, De Meyts, P, von zur Mûhlen,

A, and Ørskov, H. (1995) International symposium on glucosemetabolism and growth factors. Metabolism, 44, 1–123.

[3] Flyvbjerg, A. (2000) Putative pathophysiological role of growthfactors and cytokines in experimental diabetic kidney disease.Diabetologia, 43, 1205–1223.

[4] Flyvbjerg, A., Ørskov, H., and Alberti, KGMM (Eds). (1993)Growth Hormone and Insulin-Like Growth Factor I in Humanand Experimental Diabetes, pp. 1–322. New York, John Wiley& Sons.

[5] Hansen, Aa. P. (1972) Serum growth hormone patterns in juvenilediabetes. Dan. Med. Bull., 19, 1–32.

[6] Hansen, Aa. P., and Johansen, K. (1970) Diurnal patterns of bloodglucose, serum free fatty acids, insulin, glucagon and growthhormone in normals and juvenile diabetics. Diabetologia, 6, 27–33.

[7] Schaper, N. C. (1990) Growth hormone in type I diabetic andhealthy man. Acta Endocrinol. (Copenh.), 122, 1–47.

[8] Yde, H. (1969) Abnormal growth hormone response to ingestionof glucose in juvenile diabetics. Acta Med. Scand., 186, 499–504.

[9] Ndon, J. A., Giustina, A., and Wehrenberg, W. B. (1992) Hy-pothalamic regulation of impaired growth hormone secretion indiabetic rats. 1. Studies in streptozotocin-induced diabetic rats.Neuroendocrinology, 55, 500–505.

[10] Ortiz-Caro, J., Gonzalez, C., and Jolin, T. (1984) Diurnal varia-tions of plasma growth hormone, thyrotropin, thyroxine, and tri-iodothyronine in streptozotocin-diabetic and food-restricted rats.Endocrinology, 115, 2227–2232.

[11] Tannenbaum, G. S. (1981) Growth hormone secretory dynamicsin streptozotocin diabetes: Evidence of a role for endogenouscirculating somatostatin. Endocrinology, 108, 76–82.

[12] Bluet-Pajot, M. T., Durand, D., and Kordon, C. (1983) Influenceof streptozotocin-induced diabetes on growth hormone secretionin the rat. Neuroendocrinology, 36, 307–309.

[13] Serri, O., and Brazeau, P. (1987) Growth hormone responsivenessin vivo and in vitro to growth hormone releasing factor in thespontaneously diabetic BB Wistar rat. Neuroendocrinology, 46,162–166.

[14] Ndon, J. A., Giustina, A., and Wehrenberg, W. B. (1992) Hy-pothalamic regulation of impaired growth hormone secretion indiabetic rats. 2. Studies in spontaneously diabetic BB Worcesterrats. Neuroendocrinology, 55, 506–511.

[15] Giustina, A., and Veldhuis, J. D. (1998) Pathophysiology of theneuroregulation of growth hormone secretion in experimentalanimals and the human. Endocrine Rev., 19, 717–797.

[16] Flyvbjerg, A., Bennett, W. F., Rasch, R., Kopchick, J. J., andScarlett, J. A. (1999) Inhibitory effect of a growth hormone re-ceptor antagonist (G120K-PEG) on renal enlargement, glomeru-lar hypertrophy, and urinary albumin excretion in experimentaldiabetes in mice. Diabetes, 48, 377–382.

[17] Hossenlopp, P., Seurin, D., Segovia-Quinson, B., Hardouin, S.,and Binoux, M. (1986) Analysis of serum insulin-like growth fac-tor binding proteins using western blotting: Use of the method fortitration of the binding proteins and competitive binding studies.Anal Biochem., 154, 138–143.

[18] Flyvbjerg, A., Kessler, U., Dorka, B., Funk, B., Ørskov, H.,and Kiess, W. (1992) Transient increase in renal insulin-likegrowth factor binding proteins during initial kidney hypertro-phy in experimental diabetes in rats. Diabetologia, 35, 589–593.

[19] Flyvbjerg, A. (1997) Role of growth hormone, insulin-likegrowth factors (IGFs) and IGF-binding proteins in the renal com-plications of diabetes. Kidney Int., 60, S12–S19.

[20] Bornfeldt, K. E., Arnqvist, H. J., Enberg, B., Mathews, L. S.,and Norstedt, G. (1989) Regulation of insulin-like growth factor-I and growth hormone receptor gene expression by diabetesand nutritional state in rat tissues. J. Endocrinol., 122, 651–656.

[21] Maes, M., Ketelslegers, J. M., and Underwood, L. E. (1983)Low plasma somatomedin-C in streptozotocin-induced diabetesmellitus. Correlation with changes in somatogenic and lactogenicliver binding sites. Diabetes, 32, 1060–1069.

[22] Maes, M., Underwood, L. E., and Ketelslegers, J. M. (1986)Low serum somatomedin-C in insulin-dependent diabetes: Evi-dence for a postreceptor mechanism. Endocrinology, 118, 377–382.

GH HYPERSECRETION IN STZ DIABETIC MICE 81

[23] Massa, G., Verhaeghe, J., Vanderschueren-Lodeweyckx, M., andBouillon, R. (1993) Normalization of decreased plasma concen-trations of growth hormone-binding protein by insulin treatmentin spontaneously diabetic BB rats. Horm. Metab. Res., 25, 325–326.

[24] Johansen, P. B., Nowak, J., Skjaerbaek, C., Flyvbjerg, A.,Andreassen, T. T., Wilken, M., and Ørskov, H. (1999) Ipamorelin,a new growth-hormone-releasing peptide, induces longitudinalbone growth in rats. Growth Horm. IGF Res., 9, 106–113.

[25] Johansen, P. B., van Neck, J. W., and Flyvbjerg, A. (2000) Ef-fects of ipamorelin on GH secretion, GH receptor, GH bindingprotein, and IGF-I mRNA in mice. Growth Horm. IGF Res., 10,P128.

[26] Guillaume, V., Magnan, E., Cataldi, M., Dutour, A., Sauze, N.,Renard, M., Razafindraibe, H., Conte-Devolx, B., Deghenghi,R., and Lenaerts, V. (1994) Growth hormone (GH)-releasing hor-mone secretion is stimulated by a new GH-releasing hexapeptidein sheep. Endocrinology, 135, 1073–1076.

[27] Tannenbaum, G. S., and Bowers, C. Y. (2001) Interactions ofgrowth hormone secretagogues and growth hormone-releasinghormone/somatostatin. Endocrine, 14, 21–27.

[28] Landau, D., Segev, Y., Eshet, R., Flyvbjerg, A., and Phillip, M.(2000) Changes in the growth hormone-IGF-I axis in non-obesediabetic mice. Int. J. Exp. Diabetes. Res., 1, 9–18.

[29] Mercado, M., Molitch, M. E., and Baumann, G. (1992) Lowplasma growth hormone binding protein in IDDM. Diabetes, 41,605–609.

[30] Postel-Vinay, M. C., Cohen-Tanugi, E., and Charrier, J. (1982)Growth hormone receptors in rat liver membranes: effects offasting and refeeding, and correlation with plasma somatomedinactivity. Mol. Cell Endocrinol., 28, 657–669.

[31] Murao, S., Sato, M., Tamaki, M., Niimi, M., Ishida, T., andTakahara, J. (1995) Suppression of episodic growth hormonesecretion in streptozotocin-induced diabetic mice: Time-coursestudies on the hypothalamic pituitary axis. Endocrinology, 136,4498–4504.

[32] Chen, N. Y., Chen, W. Y., and Kopchick, J. J. (1996) A growthhormone antagonist protects mice against streptozotocin inducedglomerulosclerosis even in the presence of elevated levels of glu-cose and glycated hemoglobin. Endocrinology, 137, 5163–5165.

[33] Esposito, C., Liu, Z. H., Striker, G. E., Phillips, C., Chen, N. Y.,Chen, W. Y., Kopchick, J. J., and Striker, L. J. (1996) Inhibition ofdiabetic nephropathy by a GH antagonist: A molecular analysis.Kidney Int., 50, 506–514.

[34] Segev, Y., Landau, D., Rasch, R., Flyvbjerg, A., and Phillip, M.(1999) Growth hormone receptor antagonism prevents early renalchanges in nonobese diabetic mice. J. Am. Soc. Nephrol., 10,2374–2381.

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Disease Markers

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com